Method and kit for calibrating a photoluminescence measurement

ABSTRACT

The invention is directed to a method and a kit for calibrating a photoluminescence measurement system, in particular a fluorescence measurement system. The kit includes a number of fluorescence standards i and their corrected and certified fluorescence spectra I i (λ), whereby the fluorescence standards i are selected, so that their spectrally corrected fluorescence spectra I i (λ) cover a broad spectral range with high intensity. The standards are characterized by large half-widths FWHM i  of their bands of at least 1400 cm −1 . According to the method of the invention, partial correction functions F i (λ) are generated by forming the quotient of the measured fluorescence spectra J i (λ) and the corresponding corrected fluorescence spectra I i (λ), which are then combined to form a total correction function F(λ) for a broad spectral range. The combination factors α i  are hereby computed by statistical averaging of consecutive partial correction functions F i (λ) over only a predefined, limited overlap region λ i/i+1 ±Δλ OL  about the mutual crossover wavelength λ i/i+1 .

The invention relates to a method and a kit for calibrating aphotoluminescence measurement system, in particular a fluorescencemeasurement system. The invention also relates to the use of the kit aswell as the use of selected chemical compounds as fluorescence standardsfor calibrating photoluminescence measurement systems.

Each luminescence measurement technique provides measurement data withanalyte-specific and device-specific contributions. The undesirable,device-specific contributions reflect the wavelength- andpolarization-dependence of device components of the employed measurementdevice. These dependencies are caused, in particular, by the opticalcomponents in the excitation and emission channel of the device, theexcitation light source, and the employed detection systems. Acomparison between luminescence data from different devices andlaboratories, measurements reflecting device aging, the demand fortraceability of luminescence data to radiometric primary standards(according to the general requirement in EN ISO/IEC 17025) as well asmany quantitative fluorescence results, the determination of relativefluorescence quantum yields and the optimization of luminescence methodsrequire the determination of these device-specific contributions. Thisapplies, in particular, to a comparative evaluation of spectrallyshifted luminescence profiles or to emission measurements at differentexcitation wavelengths.

Photoluminescence measurement devices generally have an excitationchannel which can include an excitation light source and awavelength-selective optical component, and an emission channel, whichis typically arranged perpendicular to the optical path of theexcitation light and is used to record the light (photoluminescence)emitted by the chromophore located in the sample space after lightabsorption. Frequently, a defined fraction of the excitation light iscoupled with a beam splitter into a reference channel, which includes anoptical component, such as a mirror or a scatterer and a (reference)detector. The reference channel is used to record the actual excitationlight intensity at the excitation wavelength so as to capture short-termvariations in the intensity of the excitation light. The aforementioneddevice-specific contributions to the fluorescence signal can bedetermined by determining so-called correction functions, which describethe wavelength- and polarization-dependence of these effects for theemission and excitation channel of the corresponding device. Thecorrection functions are determined independent of each other. Theemission correction function includes the wavelength- andpolarization-dependent transmission efficiency of the optical componentsin the emission channel and the wavelength- and polarization-dependentspectral sensitivity of the employed detection system. The excitationcorrection function describes the wavelength-dependent spectral radianceof the excitation light source and the wavelength- andpolarization-dependent transmission efficiency of the optical componentsin the excitation channel.

It is known to employ certified physical transfer standards fordetermining the device-specific effects. Typically, certified receiverstandards are used for calibrating the excitation channel, and certifiedstandard lamps are used for calibrating the emission channel.Disadvantageously, the use of physical transfer standards requires fromthe user a thorough optical understanding of the application, expensiverecalibrations, changes in the spectral radiance of standard lamps whichdepend on their operating life, and for an emission correction withstandard lamps the different emission characteristics of lamp andsample, and the differences in spectral radiance between the transferstandard and a typical luminescent sample of potentially at least threeorders of magnitude. This can produce erroneous and substandardcorrection functions and is also complex and expensive. Physicaltransfer standards are also in many cases not suitable for thecalibration of simple, compact photoluminescence measurement systems.

So-called quantum counters can also be used for the excitationcorrection. These are highly concentrated dye solutions, whichcompletely absorb the incident light quanta and emit with awavelength-independent fluorescence quantum yield. Measurement dataobtained with quantum counters are quite dependent on concentration andgeometry, and are also susceptive to polarization effects. Standardizedcalibration methods with defined concentrations, in combination withdefined measurement geometries, are not available for quantum counters.

Also known are so-called fluorescence standards, which are typicallybased on the photoluminescence of a chemical compound. Spectralfluorescence standards or so-called emission and excitation standardswith known and corrected (for device-specific effects) emission and/orexcitation spectra can be used in a device calibration for determiningthe spectral characteristics of photoluminescence measurement systems.Such fluorescence standards are employed in various forms, in particularin the form of solutions or embedded in solid polymer or glass matrices.Fluorescence standards, in particular in the form of solutions,advantageously have a luminescence intensity and emissioncharacteristics that are very similar to that of the luminescencesamples to be investigated. Fluorescence standards therefore enable the(spectral) calibration under the conditions typical for samplemeasurements. The fluorescence standards can be measured in manydifferent types of devices, formats and measurement geometries and aretherefore also suitable for the calibration of fluorescence measurementsystems with particular sample geometries or sample formats, for examplein micro-cuvettes, micro-titer plates, or cryostat systems. Onlyfluorescence standards enable calibration in the same cuvette ormeasurement arrangement used for the actual sample measurement, so thatoptimal calibration results can be obtained. One problem withfluorescence standards is the large number of material and luminescenceproperties to be defined. A prerequisite for the suitability of atransfer standard is the complete characterization of allapplication-specific properties, including the employed methods, andinformation about the measurement uncertainty, as well as a sufficientlong-term stability in the pure solid state and in solution, or whenembedded in a matrix.

The technical literature discusses in detail many recommendationsregarding fluorescence standards, which also includes emission andexcitation standards and fluorescence quantum yield standards. Quininesulfate dihydrate (SRM936) is so far the only emission standard whosecorrected emission spectrum is certified by a government agency, in thiscase by the National Institute for Standards and Technology (NIST, USA)using a traceably characterized reference fluorometer with a knownmeasurement uncertainty (R. A. Velapoldi, K. D. Mielenz, NBS Spec. Publ.1980, 260-264, PB 80132046, Springfield, Va.). Information regarding thedye purity, the calibration of the employed spectrometer, the employedmeasurement parameters, and the measurement uncertainty is availableonly for this standard. The spectral range where fluorescence standardscan be used for calibration is limited by the position and width of thefluorescence bands. Only the band at the longest wavelength should beused for excitation standards. The emission standard quinine sulfatecovers only the spectral range from approximately 400 to 550 nm. Severalchromophores having matching fluorescence spectra must be combined forcalibrating a photoluminescence measurement system over the entireUV/vis/NIR spectral range. However, so far only a few exemplary standardcombinations are known. For example, a combination (no longercommercially available) of emission standards is known which includesfluorophore-containing polymeric foils with NIST-certified emissionspectra (A. Thommpson, K. L. Eckerle, SPIE 1989, 1054, 20, J. W.Hofstraat & M. J. Latuhidin, Appl. Spectrosc. 1994, 48, 436). The systemrequires a defined measurement geometry, the use of polarizers, andmeasurement of the luminescence in Front-Face geometry, and is thereforenot suitable for the calibration of simple measurement systems. Themeasurement conditions are also different from typical conditions forliquid samples. Combining the various partial correction functions to atotal correction function is not described.

Fluorophore-containing polymethyl-methacrylate (PMMA) blocks in the formof cuvettes are also known as emission and excitation standards. Theemployed fluorophores typically have strongly structured emission andexcitation spectra as well as steeply ascending flanks, which makes thefluorescence profile dependent on the monochromator bandpass andincreases the calibration uncertainty. Uncertainties in the wavelengthaccuracy also cause serious errors in the fluorescence intensity. Thespectra are not traceable; they do not match and cannot be combined to atotal correction function.

Other known dye solutions of different fluorophores frequently have aproblem with narrow, steeply rising emission bands and an insufficientspectral separation between absorption and emission bands. They aretherefore unsuitable as emission standards. Several substances haveinadequate photo stability and form under typical excitation andmeasurement conditions photo products with inherent spectralcontributions. Many employed substances have an exceedingly largefluorescence anisotropy, which represents an additional error source inthe calibration and requires the use of polarizers. In general, theaforedescribed spectra are not traceable, information about themeasurement uncertainty is lacking, and only in exceptional cases(quinine sulfate dihydrate) are the spectra certified by an authorizedagency. Frequently, the characterization of the application-relevantspectroscopic properties is also incomplete, and information about thedye purity is lacking in most cases.

A statistical solution trial for linking partial correction functions ofdifferent dye standards to a total correction function by applyingcounting rate statistics (Poisson statistics) is described by J. A.Gardecki and M. Maroncelli (Appl. Spectrosc. 1998, 52, 1179). Thefluorescence standards employed therein have sometimes theaforementioned deficiencies, such as steep and structured bands (α-NPO),inadequate photo stability in solution (tryptophane, coumarine 102) oran exceedingly high fluorescence anisotropy (LDS 751). Because theemployed mathematical solution trial is based on counting statistics,the method is designed only for devices operating in a photo countingmode and hence with very large absolute numbers. Application of thecounting statistics in devices operating in analog mode, where theintensities are represented by significantly smaller numerical values,causes significant artifacts in the so-called correction functions. Themethod is also designed for use by experienced fluorescencespectroscopists. Disadvantageously with this method, the emissionspectra provided by many fluorometers, as related to the reference light(forming the quotient of emission and reference), can also produceartifacts in the total correction function, when the partial correctioncurves of the individual emission standards are combined with eachother. These artifacts can occur when only small or slightly negativevalues, as in the fluorescence sidebands, enter the calculation aftercorrecting for blank values.

In summary, it can be stated that presently no calibration system existswhich enables a reliable, traceable and easily manageable calibrationover the entire spectral range of a photoluminescence measurementsystem, by using dye standards with a known uncertainty in theirapplication-relevant fluorescence properties, which satisfy allrequirements for a reproducible and traceable calibration. It istherefore an object of the present invention to provide a comprehensivecalibration system using fluorescence standards, and a calibrationmethod, which allows a simple and reproducible calibration that can betraced to primary radiometric standards (black body; cryo-radiometer),and which enables a reliable calibration of a photoluminescencemeasurement system over an extended spectral range.

The object is solved by a kit with fluorescence standards (emissionstandards and/or excitation standards) and by a method having thefeatures of the independent claims.

The method according to the invention for calibrating aphotoluminescence measurement system, in particular a fluorescencemeasurement system, includes the steps of

-   (a) measuring fluorescence spectra J_(i)(λ) of a plurality of    fluorescence standards i with the photoluminescence measurement    system to be calibrated, wherein the fluorescence standards i are    selected, so that their combined spectrally corrected fluorescence    spectra I_(i)(λ) cover a predefined spectral range in such a way,    that the fluorescence bands of the corrected, sequentially arranged    fluorescence spectra I_(i)(λ) have at least a minimum predefined    intensity at their crossover wavelengths λ_(i/i+1);-   (b) calculating combination factors α_(i) through statistical    averaging of consecutive partial correction functions F_(i)(λ),    wherein the partial correction functions F_(i)(λ) are formed by    forming the quotient of the measured fluorescence spectra J_(i)(λ)    and the corresponding corrected fluorescence spectra I_(i)(λ) of the    fluorescence standards i, and wherein the statistical averaging    takes place over a predefined, limited overlap region    λ_(i/i+1)±Δλ_(OL) about the mutual crossover wavelengths λ_(i/i+1);    and-   (c) determining a total correction function F(λ) for the predefined    spectral range by statistical combination of the partial correction    functions F_(i)(λ), which are weighted according to the combination    factors α_(i).

In the context of the present invention, the term corrected fluorescencespectrum refers to an emission or excitation spectrum obtained in atraceably calibrated luminescence measurement system under definedmeasurement conditions. The corrected fluorescence spectrum isdevice-independent as a result of the correction of the device-specificcontributions of the calibrated measurement system. Preferably, thespectrum is certified by an authorized agency (for example NIST).

The method of the invention is different from conventional approaches(Gardecki and Maroncelli, Appl. Spectrosc. 1998, 52, 1179) mainly inthat statistical averaging and/or weighting of the partial correctionfunctions for determining the combination factors α_(i), on which thedetermination of the total correction function depends, is performedexclusively for defined overlap regions of the partial correctionfunctions, i.e., only for a limited narrow spectral environment of thevarious crossover wavelengths. With the matching dye standards accordingto the invention, which have a minimum relative intensity at therespective crossover wavelengths of their fluorescence bands,advantageously only spectral contributions of the initial base spectrawith a relatively high-intensity and hence low error contributions areconsider in the statistical analysis. In particular, non-idealmeasurement spectra and their partial correction functions can also beused with the method of the invention. For example, the present methodalso produces a smooth and continuous total correction function forthose measurement spectra which, after correcting for blank values(subtracting the buffer spectrum), have negative intensity values in theregion of the spectral flanks.

According to the invention, the overlap region about the crossoverwavelengths is at most 12 nm in an adjacent region on both sides of therespective crossover point. More particularly, statistical averaging isperformed over a range of ±10 nm, preferably of only ±8 nm, about eachcrossover wavelengths. This feature, together with the required minimumintensities of the spectra at the crossover wavelengths, guarantees thatonly relatively high intensities are applied in the statisticalanalysis, thereby eliminating artifacts in the correction functions.

Moreover, the relative minimum intensity to be maintained at each of theindividual crossover wavelengths is at least 20% of the maximumintensity of the fluorescence bands. Less erroneous results are obtainedwith an intensity of at least 25%, preferably of at least 30% of themaximum in-band intensity. In actual applications, these intensities areapproximately 40% of the maximum in-band intensity. The aforementionedvalues apply to the UV/vis spectral range with λ≦700 nm. Because the NIRrange with λ>700 nm generally has lower quantum yields and thereforealso lower absolute intensities, lower minimum intensities at thecrossover points of at least 10%, in particular of at approximately 12%,of the maximum intensity of the respective flanking bands can betolerated in this region. In view of the limited overlap region wherethe combination factors α_(i) are statistically determined, onlyintensities of the corrected or measured (normalized) spectra of atleast 10%, in particular of at least 15%, preferably of at least 20%,relative to the maximum intensities of the corresponding spectra enterin the statistical analysis at least in the UV/vis region.

According to a particular advantageous embodiment of the method, thefunctional dependence of the total correction function in a predefined,limited spectral combination range λ_(i/i+1)±Δλ_(LK) about the crossoverwavelengths, which is narrower than the aforementioned overlap regionλ_(i/i+1)Δλ_(OL), is calculated through statistical averaging of theweighted, consecutive partial correction functions. On the other hand,the functional dependence of the total correction function outside thepredefined combination region about the crossover wavelengthscorresponds to the functional dependence of the weighted, but notaveraged, partial correction functions. Advantageously, the limitedcombination region about the crossover wavelengths is very narrow, inparticular not more than λ_(i/i+1)±5 nm, more particularly ±2 nm. Mostpreferably, the combination region includes only the correspondingcrossover point itself. In other words, the total correction function iscomposed of the weighted partial correction functions, whereby theweighted partial correction functions are averaged only at the crossoverwavelengths. As a result, the total correction function is formed onlyfrom regions of high luminescence intensity in the underlying measuredand corrected partial spectra. Intensities below the minimum intensitiesrequired at the crossover points are mostly disregarded. Excluding thosespectral segments that have a low luminescence intensity from theaverage values results in smooth total correction functions with aminimum measurement uncertainty.

According to an advantageous embodiment of the method, the totalcorrection function is formed over a predefined spectral range whichcovers at least in part the UV/vis/NIR spectral range. This predefinedspectral range extends for the emission correction from 310 to 730 nm,in particular from 300 to 950 nm, and for the excitation correction of250 to 630 nm, in particular from 240 to 900 nm. Taking into account therequirements according to the invention for the dye standards, thisrequires a combination of between 5 and 7 dyes.

According to another advantageous embodiment of the invention, astatistical, wavelength-dependent uncertainty for the devicecharacteristics and, optionally, also for the fluorescence measurementsfor the predefined spectral range of the total correction function canbe calculated by a user. This requires knowledge of thewavelength-dependent measurement uncertainty contributions for eachcorrected dye fluorescence spectrum. For example, the user can determinethe contribution of the device-specific measurement uncertainty(standard deviation) from several repeat measurements (e.g., N≧7) of thedye spectra. A total measurement uncertainty contribution for thephotoluminescence measurement system to be calibrated can then begenerated from the aforementioned wavelength-dependent measurementuncertainty contributions for the corrected spectra of the dyes and fromthe repeat standard deviation.

The kit according to the invention, which may include a set of emissionand excitation standards, for traceable calibration of thephotoluminescence system, in particular a fluorescence measurementsystem, includes the following components:

-   -   a plurality of fluorescence standards i, and    -   corrected fluorescence spectra I_(i)(λ) of the fluorescence        standards i in computer-readable form and/or a reference to an        Internet page, from which the corrected fluorescence spectra        I_(i)(λ) can be downloaded, wherein the fluorescence standards i        are selected so that their combined spectrally corrected        fluorescence spectra I_(i)(λ) cover a predefined spectral range        in such a way, that fluorescence bands of the corrected,        consecutive fluorescence spectra I_(i)(λ) have in the vis/NIR        spectral range with λ≦700 nm at least a minimum predefined        intensity at their overlapping wavelengths λ_(i/i+1) of at least        20% of the corresponding maximum in-band intensity, and (in the        same spectral range) their fluorescence bands have a respective        half-width (FWHM) of at least 1400 cm⁻¹.

The composition of the kit(s) according to the invention enables a userto reliably, reproducibly and cost-effectively calibrate the measurementsystem over a wide spectral range (310 to 730 nm, in particular from 300to 950 nm for the emission correction, and from 250 to 630 nm, inparticular from 240 to 900 nm for the excitation correction). Inparticular, matching the individual fluorescence standards according tothe invention, i.e., their half-widths as well as the required minimumintensity at the crossover wavelengths, makes it possible to generate atotal correction functions for the device with a quality that has beenunattainable to date.

According to a preferred embodiment of the invention, the kit caninclude a program algorithm for computing partial correction functionsF_(i)(λ) which executes the method steps of the aforedescribed method ofthe invention and/or a reference to an Internet page, from which thealgorithm can be downloaded.

Advantageously, the kit can include for each corrected fluorescencespectrum a device-independent, spectral measurement uncertainty curve incomputer-readable form. Optionally or in addition, a reference to anInternet page can be provided, from which the measurement data togetherwith the spectral measurement uncertainty curves can be downloaded. Withthe latter, a total measurement uncertainty curve can be generated forthe total correction function, so that the wavelength-dependentmeasurement uncertainty of the luminescence measurement system to becalibrated can be determined.

The kit may further include instructions for using the kit componentsand/or a reference to an Internet page, from which the instructions canbe downloaded. The information can include, for example, informationrelating to the measurement conditions and the device settings and thelike.

The fluorescence standards of the kit are measured in solution, becausecalibrations can then be performed under routine measurement conditions.In principle, the kit may contain the dyes in solid form or in form ofprepared solutions. The kit may also include the solvents to be used,whereby preferably the same high-purity solvent is used with alltransfers standards.

In additional embodiments of the invention, all fluorescence standardsnot only satisfy the required minimum intensity of the correctedfluorescence spectra at their respective crossover wavelengths in theUV/vis spectral range of at least 20%, in particular of at least 25%,preferably all the at least 30% of the maximum intensity of theluminescence bands (in the NIR range at least 10%, in particular atleast 12%), and the required minimum half-width, which is in particularat least 1600 cm⁻¹, preferably at least 2000 cm⁻¹, and ideally at least2400 cm⁻¹ (above 700 nm 1200 cm⁻¹, in particular 1400 cm⁻¹), but alsosatisfy the requirements described below. According to a particularlyadvantageous embodiment of the invention, all dye standards are selectedso that their fluorescence bands have a smooth and unstructured curveshape, i.e., with a spectral resolution of 1 nm, the bands have only onemaximum, no shoulders, and a continuous band waveform. Like the requiredminimum half-width associated with a small slope at the flanks of thebands, the unstructured and smooth shape of the bands also guaranteesthat the measured spectra are independent of the measurement conditionsand the device features and/or the parameter settings of the employedspectrometer, in particular the monochromator bandpass and slit width ofthe measurement channel. The band characteristics of the inventiontherefore increase the calibration reliability.

In addition, the provided fluorescence standards have a purity of atleast 98%, in particular of at least 99.5%. The overlap of excitationand emission bands of the dye standards is small. In particular, thespectral separation between absorption and emission bands is at least2000 cm⁻¹, in particular of at least 2400 cm⁻¹, and ideally at least2800 cm⁻¹. The anisotropy of the fluorescence of the dye standards in atemperature range of 20° C. to 30° C. and in the solvent to be used isin the UV/vis spectral range with λ≦700 nm at most 0.05 and preferablyat most 0.04, and in the NIR spectral range at most 0.07 and preferablyat most 0.06. Additional error sources in the calibration can beeliminated if the fluorescence spectra of the proposed dye standardshave only a small temperature dependence in the temperature range of 20°C. to 30° C. The dye standards are further characterized by a highthermal and photo-chemical stability of the pure substances and also oftheir solutions. Finally, the dyes do not form photo products undermeasurement and excitation conditions typical for staticphotoluminescence measurements. In particular, they show a maximum of10%, preferably a maximum of 2%, decrease of the fluorescence bandsafter a five-hour irradiation with light from a 100 watt xenonhigh-pressure lamp in the range of the longest wavelength absorptionmaximum at a width of the bandpass of approximately 15 nm, so that thethermal and photo-chemical stability is sufficient for the plannedapplications. In particular, the totality of the aforementionedspectroscopic and photo-chemical properties of the fluorescencestandards guarantees a high calibration reliability and traceabilityaccording to EN ISI/IEC 17025.

The kit can include a number of different emission standards and theirspectrally corrected emission spectra for generating a total correctionfunction for the emission channel (emission correction function), aswell as a number of different excitation standards and their spectrallycorrected excitation spectra for generating a total correction functionfor the excitation channel (excitation correction function). The kitpreferably includes both emission and excitation standards as well andtheir spectrally corrected emission and excitation spectra in electronicform on a data carrier or downloadable via the Internet.

More particularly, a set of preferred emission standards includecompound selected from the group consisting of biphenyl, naphthalene,coumarine, oxazin, merocyanin, hemicyanin and styryl derivates.Excitation standards are preferably selected from the group consistingof biphenyl, terphenyl, oxadizol, coumarine, oxazin, merocyanin,hemicyanin and styryl derivates. In the following, several explicitemission and/or excitation standards are listed which satisfy theaforedescribed criteria.

A first preferred emission and/or excitation standard is a biphenylderivate according to the general formula

wherein the moieties R₁ to R₁₀ independently represent a hydrogenmoiety, an alkyl or alkoxy moiety, or partially in combination with eachother an anellated, saturated hetero- or homo-nuclear ring. Preferably,R₁ and R₆ each represent an alkoxy moiety, and the remaining moietiesindependently represent a hydrogen or an alkyl moiety, wherein thealkoxy and alkyl groups independently are cyclic or acyclic, branched orlinear. Most preferably, R₁ and R₆ each represent a mesoxy moiety, andthe other moieties each represent a hydrogen moiety. This compound ispreferably used as an emission standard and has, for example, in ethanolan emission band from 290 to 410 nm at an excitation wavelength of 280nm. The half-width is approximately 4250 cm⁻¹ and the separation betweenexcitation and emission maxima approximately 5410 cm⁻¹.

A second preferred emission and/or excitation standard is a naphthalenederivate having the general formula 2

wherein the moieties R₁ to R₈ independent from each represent a hydrogenor alkoxy moiety, or partially in combination with each other ananellated, saturated, hetero- or homo-nuclear ring. Preferably, at leastone of the two naphthalene rings is substituted by two alkoxy moietiesin a mirror-symmetric position (for example, R₁ and R₄ and/or R₂ andR₃), with the remaining moieties each being hydrogen. The alkoxy groupsare acyclic, branched or linear, and do not have hydrogen moieties inβ-position relative to the ether oxygen atom. Most preferably, R₁ and R₄independently are a methoxy or neopentyloxy moiety, and the othermoieties are each hydrogen moieties. This compounds is preferably usedas an emission standard and have, for example, in ethanol an emissionband from approximately 330 to 500 nm at an excitation wavelength of 320nm. The half-width is approximately 4400 cm⁻¹ and the separation betweenexcitation and emission maxima approximately 4930 cm⁻¹.

A third preferred emission and/or excitation standard is a coumarinederivate having the general formula 3

wherein the moieties R₁ to R₇ independently represent a hydrogen moietyor an unsubstituted or a substituted alkyl moiety, or partially incombination with each other an anellated, saturated homo-nuclear ring.R₁ and R₂ independently represent a hydrogen moiety or an unsubstitutedor a substituted alkyl moiety, or an anellated, saturated homo-nuclearring, R₃ to R₅ each a hydrogen moiety, and R₆ and R₇ independently ahydrogen moiety or an alkyl moiety. Preferably, R₁ to R₅ each representa hydrogen moiety and R₆ and R₇ each an ethyl group. This compound isused as an emission and/or excitation standard and has, for example, inethanol an emission band at approximately 400 to 600 nm at an excitationwavelength of 380 nm, as well as an excitation band of 325 to 430 nm ata detection wavelength of 460 nm. The half-width of the emission band isapproximately 2850 cm⁻¹ and the separation between excitation andemission maxima approximately 4340 cm⁻¹. The excitation band has ahalf-width of approximately 3780 cm⁻¹.

A fourth preferred emission and/or excitation standard is a coumarinederivate having the general formula 4

wherein the moieties R₁ and R₂ independently represent a hydrogen moietyor an unsubstituted or a substituted alkyl moiety, or in combinationwith each other an anellated, saturated homo-nuclear ring, with n=1 or2. Preferably, R₁ represents a hydrogen and R₂ an unsubstituted or asubstituted alkyl moiety, with n=1 or 2. Most preferably, R₁ is ahydrogen moiety and R₂ a trifluoromethyl moiety, with n=2. This compoundis used as an emission and/or excitation standard and has, for example,in ethanol an emission band at approximately 460 to 700 nm at anexcitation wavelength of 420 nm, as well as an excitation band of 330 to490 nm at a detection wavelength of 530 nm. The half-width of theemission band is approximately 2890 cm⁻¹ and the separation betweenexcitation and emission maxima approximately 4940 cm⁻¹. The excitationband has a half-width of approximately 4010 cm⁻¹.

A fifth emission and/or excitation standard is an oxazin derivate, inparticular a 3H-phenoxazin-5-on derivate having the general formula 5

wherein the moieties R₁ and R₂ independently represent an unsubstitutedor a substituted alkyl moiety. Preferably, R₁ and R₂ each represent anunsubstituted linear alkyl moiety, in particular an ethyl moiety. Thecompound is used as an emission and/or excitation standard and has, forexample, in ethanol an emission band at approximately 570 to 750 nm atan excitation wavelength of 550 nm, as well as an excitation band of 440to 630 nm at a detection wavelength of 630 nm. The half-width of theemission bands is approximately 1630 cm⁻¹ and the separation betweenexcitation and emission maxima approximately 2440 cm⁻¹. The excitationband has a half-width of approximately 2960 cm⁻¹.

A sixth emission and/or excitation standard is a styryl derivate havingthe general formula 6

wherein R₁ and R₂ independently represent an unsubstituted or asubstituted alkyl moiety, R₃ a hydrogen moiety or an unsubstituted or asubstituted alkyl moiety. The C atoms designated with an asterisk can beindependently bridged by a saturated C5 or C6 ring, as long as they arein a relative 1,3-position. Preferably, R₁ to R₃ are each a methylmoiety, and no bridging is present. This compound is preferably used asan emission standard and has, for example, in ethanol an emission bandat approximately 530 to 750 nm at an excitation wavelength of 460 nm.The half-width of the emission band in acetone is 2323 cm⁻¹ and theseparation between excitation (λ_(ex)=462 nm) and emission maxima(λ_(max)=626 nm) is 5670 cm⁻¹.

An additional emission and/or excitation standard is a styryl derivate,in particular a hemicyanin derivate, having the general formula 7

wherein R₁ to R₃ independently represent an unsubstituted or asubstituted alkyl moiety and X⁻ an arbitrary anion. The C atomsdesignated with an asterisk can be independently bridged by a saturatedC5 or C6 ring, as long as they are in a relative 1,3-position.Preferably, R₁ is an ethyl moiety, R₂ and R₃ are each a methyl moiety,and X⁻ a perchlorate anion, and no bridging is present. This compound ispreferably used as an emission standard and has an emission band atapproximately 600 to 800 nm at an excitation wavelength of 500 nm. Thehalf-width of the emission band in acetone is 2808 cm⁻¹ and theseparation between excitation (λ_(ex)=492 nm) and emission maxima(λ_(max)=719 nm) is 6417 cm⁻¹.

Another emission and/or excitation standard is a styryl derivate, inparticular a hemicyanin derivate, having the general formula 8

wherein R₁ to R₅ independently represent a hydrogen moiety or anunsubstituted or a substituted alkyl moiety and X⁻ an arbitrary anion.The C atoms designated with an asterisk independently can be bridged bya saturated C5 or C6 ring, as long as they are in a relative1,3-position. Preferably, R₁ to R₅ are each a methyl group and X⁻ aperchlorate anion.

This compound is preferably used as an emission standard and has anemission band at approximately 700 to 920 nm at an excitation wavelengthof 580 nm. The half-width of the emission band in acetone is 1460 cm⁻¹and the separation between excitation (λ_(ex)=564 nm) and emissionmaxima (λ_(max)=810 nm) is 5380 cm⁻¹.

Another preferred emission and/or excitation standard is p-terphenylhaving the general formula 9

wherein R₁ to R₃ independently represent hydrogen, or an alkyl or alkoxymoiety, preferably however hydrogen. The compound is preferably used asan excitation standard and has an excitation band, for example inethanol, at approximately 240 to 320 nm if the fluorescence is detectedat 335 nm. The half-width of the excitation band is 5580 cm⁻¹.

Another preferred emission and/or excitation standard is a1,3,4-oxadiazol derivate having the general formula 10

wherein R₁ to R₄ independently represent hydrogen, or an alkyl or alkoxymoiety, preferably however hydrogen. This compound is preferably used asan excitation standard and has an excitation band, for example inacetonitrile, at approximately 275 to 350 nm and an emission maximum atabout 373 nm. The half-width of the excitation band is 4880 cm⁻¹.

According to a preferred embodiment, a kit includes a set of emissionstandards, which includes fluorescence emission standards according toeach of the general formulas 1 to 5. Such kit covers the spectral rangeof approximately 310 to 730 nm. In addition, the calibration kit caninclude at least one additional emission standard, selected from thegeneral formulas 6, 7 and 8, for the emission channel, whereby a totalcorrection function for the range from approximately 300 to 950 nm canbe generated.

In particular preferred kit for generating a total correction functionfor the excitation channel includes a set of excitation standards, withthe set including excitation standards according to each of the generalformulas 3, 4, 5, 9 and 10.

None of the compounds of the general formulas 1, 2, and 5 to 10 havebeen used to date as fluorescence standards (emission and/or excitationstandards), in particular not in the context of matching dye sets.

The kit of the invention and/or its dye standards can not only be usedfor the aforedescribed spectral calibration of photoluminescencemeasurement devices, but also as fluorescence quantum yield standardsfor determining relative fluorescence quantum yields in the UV/vis/NIRspectral range, i.e., for a quantitative calibration of the intensities.The fluorescence quantum yield is predefined as the ratio between thenumber of photons emitted by a sample and the number of photons absorbedby the sample. The kit and/or its dye standards may also be used todetermine the linear region of a detection system of a photoluminescencemeasurement system. A detector range is hereby determined where theindicated intensity increases linearly with the incident intensity,i.e., where a reliable quantitative statement about a concentration of achromophore in the probe is possible.

Additional advantageous embodiment of the invention are recited in theother dependent claims.

Embodiments of the invention will be described in more detail below withreference to the drawings. It is shown in:

FIG. 1 emission and excitation spectra of a set according to theinvention with five emission standards;

FIG. 2 emission bands of the emission standards C and D from FIG. 1;

FIG. 3 a process flow diagram of the method of the invention forcalibrating a luminescence spectrometer;

FIG. 4 diagrams of the corrected (certified) and the measured emissionspectra of the dye standards A to E, as well as of the partialcorrection functions and of the total correction function;

FIG. 5 application of the total correction function to a fluorescencespectrum of quinine sulfate; and

FIG. 6 application of the total correction function to a fluorescencespectrum of DCM.

In the present example, the emission channel of a luminescencemeasurement system, here a fluorescence spectrometer (fluorometer), iscalibrated with a set of five fluorescence standards (emissionstandards) A, B, C, D and E, wherein A is a biphenyl, B a naphthalene, Cand D each a coumarine, and E an oxazin derivate. The actual exampleincludes the following dyes:

All dyes have a purity of better than 99%, and are stable over extendedperiods of time in solid form when stored in the dark at 4° C. in thepresence of ambient oxygen. The air-saturated solutions are sufficientlystable over extended periods of time when stored in the dark at 4° C.and do not form photo products, which absorb or emit in the spectralrange intended for calibration, under the measurement and excitationconditions typically employed in photoluminescence. FIG. 1 shows thespectrally corrected and normalized emission spectrum of the emissionstandards A to E (top) and the corrected and normalized excitationspectrum of the five excitation standards AX to EX. As can be seen, allstandard spectral bands have a broad and unstructured curve shape withonly one maximum and without shoulders or discontinuities in thespectral range used for calibration. In addition, the fluorescence dyeswere selected with emphasis on the greatest possible wavelengthseparation between the maxima of the emission spectra (Em) and themaxima of the associated absorption spectra (Abs) (Stokes shift) so asto prevent re-absorption of emitted photons due to the overlap betweenthe EM and Abs bands. In particular, the separation for the dyes A, B,C, D, and E is approximately 5400, 4900, 4300, 4900, and 2400 cm⁻¹,respectively. The dyes also have a small anisotropy with r≦0.05 (UV/visspectral range with λ≦700 nm) as well as fluorescence bands with asufficiently small temperature dependence in a temperature range between20° C. and 30° C.

FIG. 2 shows additional important characteristics of the selected dyeswith reference to the exemplary corrected and normalized emissionspectra of the fluorescence standards C and D. As can be seen, theintensity I(λ_(C/D)) of the two spectra I_(C)(λ) and I_(D)(λ) at theircrossover point λ_(C/D) is greater than 20% of the corresponding maximum(normalized) intensity I_(C)(λ_(max)) and I_(D)(λ_(max)). This conditionis satisfied also for all the other crossover wavelengths) λ_(i/i+1).For the exemplary compounds A to E, all the crossover points have anintensity of at least 40% of the maximum intensities. The half-widthFWHM_(D) is indicated for the dye D, which according to the invention isgreater than 1400 cm⁻¹ at least in the UV/vis spectral range for alldyes. The half-widths of the emission spectra of the exemplary compoundsA to E are approximately 4250, 4400, 2850, 2890, and 1630 cm⁻¹,respectively.

The spectral range of the described combination of standards can beextended into the NIR spectral range by including additional dyes, forexample the aforementioned merocyanine and styryl compounds according tothe aforedescribed general structures 6, 7 and 8. These are subject tosimilar selection criteria and requirements, wherein in the spectralrange at λ>700 nm only a minimum intensity at the overlap points of, inparticular, at least 12% of the maximum intensity, a half-width of inparticular at least 1200 cm⁻¹, and an anisotropy of in particular r≦0.07is required.

The process flow for the calibration method according to the inventionis shown in FIG. 3 in conjunction with FIG. 4. Starting at step S1 ofFIG. 3, the raw emission spectra J_(i) ^(R)(λ) of the five (or more) ofthe aforedescribed fluorescence standards A to E included in the kit aremeasured with the measurement system to be calibrated. The measurementconditions at the photoluminescence measurement device—such as slitwidth, detector voltages, scan mode and scan speed, filters, polarizersand polarization angles, etc., are set to the parameters typically usedto record fluorescence spectra and/or for which spectrally correctedluminescence spectra are required. A spectrum of blank values J_(i)^(B)(λ) of the employed solvents is also measured under the samecondition as the dye measurement.

The following user-friendly computation of a total correction functionfor the fluorometer is performed with a program algorithm, whichperforms the subsequent steps. In the following step S2, the measuredraw spectra J_(i) ^(R)(λ) and the blank value spectra J_(i) ^(B)(λ) areread by the program. The program expects at least two or more measuredfluorescence spectra of spectrally consecutive dyes and of theassociated spectra of the blank values in an ASCII-DAT or -CVS format.The program supports spectral step intervals Δλ(J_(i)) between themeasurement points from 0.1 to 10 nm, with step widths between 0.5 and2.0 being preferred.

In step S3, the read-in spectra are analyzed by different subroutines.The subroutines include, for example, a check of the formatcompatibility of the spectra and optionally a format conversion so thatthey can be read by the program, sorting with respect to the wavelengthrange and association with a dye group or with the group of blankvalues. In addition, in this step, all spectral ranges havingintensities lower than an intensity threshold of 5%, relative to theassociated maximum intensity, are clipped. Another routine associates ofthe measured spectra J_(i)(λ) with the corresponding certified spectraI_(i)(λ).

In step S4, the blank values are corrected by subtracting the solventspectra J_(i) ^(B)(λ) from the respective measured spectra J_(i)^(R)(λ), thereby generating the blank-value-corrected measured spectraJ_(i)(λ). Blank values are corrected only if the routine determines thatall spectral limits (λ_(Start) and λ_(Ende)) of the dye spectra J_(i)^(R)(λ) are identical to those of the blank value spectra J_(i) ^(B)(λ).Otherwise, the program assumes that the blank values were alreadycorrected by the user and enters a corresponding remark into the logfile.

In step S5, the spectrally corrected and certified spectra I_(i)(λ) forthe five fluorescence standards together with their combineduncertainties are then read from a binary file. The spectra I_(i)(λ)were measured with a traceably calibrated fluorescence spectrometer witha known measurement uncertainty, were corrected for blank values andother spectral features, and are therefore now device-independent andtraceable to the radiometric primary standard “black body” or“cryo-radiometer.” Because error reports were generated for allindividual steps used for obtaining the fluorescence curves and for theapplied certificates, a combined wavelength-dependent uncertainty orerror of the measured intensities can be provided for the fluorescencestandards.

In step S6, the scan step length Δλ_(I) of the certified spectraI_(i)(λ) is matched, if required, to the scan step length Δλ_(i) of themeasured spectra J_(i)(λ). The missing intermediate values I_(i)(λ_(k))between each set of eight consecutive data points (k−3 . . . k . . .k+4) of the fluorescence standards are interpolated with a (smaller)spacing Δλ_(I) by using a third-degree polynomial function (spline). Thespline window is then moved sequentially across all points of eachspectrum I_(i)(λ). As a result, the certified spectra I_(i)(λ) and thespectra J_(i)(λ) corrected for the blank values have the same basis Δλ.

The lower part of FIG. 4 shows curves obtained by calibration on aPerkin-Elmer LS 50 B fluorometer for the blank-value-corrected emissionspectra J_(i)(λ) (broken lines) and the certified emission spectraI_(i)(λ) after intensity normalization (continuous lines). Thereafter,in step S7, according to FIG. 3 the signal-to-noise ratio S/N of themeasured spectra J_(i)(λ) is analyzed over a spectral range limited tothe vicinity of the maxima. The statistical data quality is important,since the noise of the measured curves is directly transferred to thespectral correction function to be generated and therefore enters thespectral correction of each measured spectrum.

Parameters for the polynomial equations up to the 9^(th) degree areestimated by solving the matrices for all measured spectra J_(i)(λ) inthe region of their maxima (flank intensity 97 . . . 100 . . . 97%). Foreach dye i, the polynomial with the smallest deviation from themeasurement data (least-square error) is assumed to be the “noise-freetrue curve” for the spectrum. The inverse standard deviation of themeasurement points from the respective best polynomial curve isconsidered as a measure of the noise in the region about thecorresponding band maximum. If the measure of noise is below theempirically predefined value of, for example, 1000, then a suggestion isnoted in the log file, advising the user to repeat the measurement ofthe corresponding chromophore a number of times in order to arrive at atotal correction function with a sufficiently low scatter.

The program subsequently goes to step S8, where the crossover points(more accurately: intersecting wavelengths)λ_(i/i+1) of respectivespectrally consecutive emission spectra are determined (see FIG. 2). Thecrossover point λ^(I) _(i/i+1) (or λ^(J) _(i/i+1)) between two spectrais the wavelength, where the intensity of the normalized preceding(decreasing) spectrum is closest to the normalized following(increasing) spectrum, i.e., where the curves cross over. The crossoverpoint λ_(i/i+1) (or λ_(A/B)) is determined and averaged for respectiveintersecting pairs of spectra both for the measured spectraJ_(i)(λ)/J_(i+1)(λ): λ^(J) _(i/i+1) (e.g., J_(A)(λ), J_(B)(λ)) and forthe corresponding certified spectra: λ^(I) _(i/i+1) (e.g., I_(A)(λ),I_(B)(λ)). The crossover point λ_(i/i+1) of both pairs is determined asthe closest rounded wavelength value which exists in all four adjacentspectra, as an average from the two pair average values λ^(I) _(i/i+1)and λ^(J) _(i/i+1).

In step S9, combination factors α_(i) are determined for each dye i bystatistical averaging of mutually overlapping spectral ranges in theregion±Δλ about the respective crossover wavelength λ_(i/i+1) of allfour adjacent spectra I_(i)(λ), I_(i+1)(λ), J_(i)(λ), and J_(i+1)(λ)with the equations 1 to 3. In this step, the partial correctionfunctions F_(i)(λ) are implicitly computed with equation 2 by formingthe quotient between the corrected spectra I_(i)(λ) and thecorresponding measured spectra J_(i)(λ) for the individual fluorescencestandards i. The curves of the (unweighted) partial correction spectraF_(A)(λ) to F_(E)(λ) for the standards A to E are illustrated in the topsection of FIG. 4.

$\begin{matrix}{\alpha_{i + 1} = \frac{\sum\limits_{\lambda}\left\lbrack {\frac{F_{i}(\lambda)}{F_{i + 1}(\lambda)}/{\sigma_{{i/i} + 1}^{2}(\lambda)}} \right\rbrack}{\sum\limits_{\lambda}{1/{\sigma_{{i/i} + 1}^{2}(\lambda)}}}} & (1) \\{{F_{i}(\lambda)} = \frac{I_{i}(\lambda)}{J_{i}(\lambda)}} & (2) \\{{\sigma_{{i/i} + 1}^{2}(\lambda)} = {\left\lbrack {\frac{1}{J_{i}(\lambda)} + \frac{1}{J_{i + 1}(\lambda)}} \right\rbrack \cdot \left\lbrack \frac{F_{i}(\lambda)}{F_{i + 1}(\lambda)} \right\rbrack^{2}}} & (3)\end{matrix}$

For the first combination factor, α₁≡1. The summand in equation 1 forthe combination factor α_(i) is determined only over a predefinedoptimized spectral overlap region±Δλ_(OL) about the respective crossoverwavelengths λ_(i/i+1). In the present example, statistical averaging fordetermining α_(i+1) according to equation 1 is performed over a regionof ±8 nm adjacent on both sides of the crossover wavelengths λ_(i/i+1).For example, if the crossover point λ_(A/B) is located at 350 nm, thenα_(B) is determined by statistical averaging of the partial correctionfunctions F_(A)(λ) and F_(B)(λ) over the spectral range 350±8 nm.

In step S10, the connection factors β_(i) are determined for eachfluorescence standard i according to equation 4 by multiplying allspectrally preceding combination factors α_(i). α₁≡1 requires β₁=1.

$\begin{matrix}{\beta_{i} = {\prod\limits_{k = 1}^{i}\alpha_{k}}} & (4)\end{matrix}$

Finally, in steps S11 to S13, the total correction function F(λ) iscomputed. Although according to the employed program algorithm thesesteps do not represent separate steps (but rather a single computingstep consisting in the summation according to equation 5), steps S11 toS13 are shown in FIG. 3 as separate steps for a better understanding.Accordingly, in step S11, the values of the function F(λ) are initiallydetermined in a predefinable combination region λ_(i/i+1)±Δλ_(LK) aboutthe respective crossover points λ_(i/i+1) according to equations 5 and6. In the combination region λ_(i/i+1)±Δλ_(LK), the overlapping partialcorrection functions F_(i)(λ), which are weighted by β_(i), arestatistically averaged. In the present example, equation 5 is appliedonly to the crossover wavelengths λ_(i/i+1) determined in step S8, i.e.,Δλ_(LK)=0, according to a predetermined execution mode of the program.

$\begin{matrix}{{{F(\lambda)} = \frac{\sum\limits_{i = 1}^{N}{\beta_{i} \cdot {{F_{i}(\lambda)}/{\sigma^{2}(\lambda)}}}}{\sum\limits_{i = 1}^{N}{1/{\sigma^{2}(\lambda)}}}}{with}} & (5) \\{{\sigma^{2}(\lambda)} = {\frac{\left\lbrack {\beta_{i}{F_{i}(\lambda)}} \right\rbrack^{2}}{J_{i}(\lambda\;)}.}} & (6)\end{matrix}$

All other points of the correction function F(λ), i.e., in the regions),λ≠λ_(i/i+1)±Δλ_(LK), are according to the predefined execution mode ofthe program not determined in step S12, but are simply computedaccording to equation 7. (This is the same as a summing in equation 5over only one i). In other words, the total correction function F(λ)outside the combination regions λ_(i/i+1)±Δλ_(LK), in particular for allwavelengths outside the crossover points λ_(i/i+1), corresponds to thepartial correction functions F_(i)(λ) weighted with the factors β_(i)determined for these functions F_(i)(λ).F(λ)=β_(i) ·F _(i)(λ)  (7)

The effective range of the equations 5 and 6 in the program canoptionally be extended to broader spectral combination regionsλ_(i/i+1)±Δλ_(LK) about the crossover wavelengths through user input ofcorresponding control variables. The exemplary dataset in FIG. 4 (top)shows that this is frequently not advantageous, since the partialcorrection functions F_(C)(λ) and F_(D)(λ) significantly deviate attheir respective starting regions and the partial quotient F_(E)(λ)significantly deviates at the end from otherwise continuous curveshapes. The program was therefore used in default mode for the emissioncorrection function F(λ), which is also shown at the top of FIG. 4.Stated differently, only the filled parts of the individual quotientsF_(i)(λ) between the crossover wavelengths λ_(i/i+1) were computed withthe connection factors β_(i) according to FIG. 7. An average between thetwo adjacent partial quotients was formed at the intersections λ_(i/i+1)according to equation 5, yielding the total correction function F(λ)(black line), which is smooth and continuous. Because α₁=1 and β₁=1,F(λ) corresponds up to the first crossover point λ_(A/B) to the partialcorrection function F_(A)(λ) weighted with β_(A).

In step S13, the values of the concatenated total correction functionF(λ) are outputted as an ASCII table in the file “CorrF.TXT”, and allactions and error messages of the program are documented in a log file“LINKLOG#.TXT.”

All spectra measured with a luminescent spectrometer calibrated in thismanner and with the calibration settings are spectrally corrected, aftersubtraction of corresponding blank value spectra, by simplemultiplication with a correction function F(λ). Values of the measuredspectrum and the spectral correction function F(λ) must only bemultiplied with each other, if they are associated with the samewavelength, i.e., the same base Δλ. In this way, traceable, spectrallycorrected luminescent spectra expressed in relative intensity units canbe obtained.

The aforedescribed example is related to the generation of a totalcorrection function for the emission by using emission standards. Aspectral excitation correction function can be obtained by measuring aset of excitation standards with a luminescence spectrometer in the samemanner and by computing the obtained excitation spectra with a datasetfor the spectrally corrected (certified) excitation spectra of thefluorescence standards according to the program flow in FIG. 3.

FIG. 5 shows in form of an example, how the total correction functionF(λ) obtained according to FIGS. 3 and 4 can be applied to thefluorescence of quinine sulfate. The continuous curve shows thespectrally uncorrected emission spectrum J(λ) measured with afluorometer (SPEX Fluorolog) within excitation wavelengths of 348 nm.The spectrum was, however, corrected for blank values. The spectrallycorrected emission spectrum (dotted curve) is obtained aftermultiplication with the emission correction function F(λ) obtained forthe fluorometer by using the fluorescence standard kit according to theinvention. Unlike the spectrally uncorrected spectrum, the spectrallycorrected emission spectrum lies in all regions below the error limitsdefined by NIST (gray triangles).

FIG. 6 clearly shows the potential impact of the spectral correction onthe spectral position on the fluorescence maximum. The raw emissionspectrum J(λ) of DCM in acetone nitrile was recorded on a fluorometer LS50B from the company Perkin Elmer. The spectrally corrected spectrum(dotted curve), which is bathochrome shifted by 11 nm, is obtained byapplying the spectral emission correction (multiplication of themeasured raw spectrum J(λ) with the emission correction function F(λ)).This curve deviates only slightly from the spectrum measured with afluorometer SLM 8100, which had been spectrally corrected with anemission correction function F(λ)_(tr) produced with a radiance transferstandard (gray curve).

LIST OF REFERENCE SYMBOLS/ABBREVIATIONS

-   i consecutive numbering of a fluorescence standard in the kit with    1≦i≦N-   I_(i)(λ) spectrally corrected (certified) fluorescence spectrum of    the fluorescence standard i-   J_(i)(λ) measured, blank value corrected fluorescence spectrum-   F_(i)(λ) partial correction function of the fluorescence standard i-   F(λ) total correction function-   λ_(i/i+1) crossover wavelengths of consecutive spectra of the i^(th)    and the (i+1)^(th) standard-   α_(i) combination factor of adjacent partial correction functions-   β_(i) connection factor-   λ_(i/i+1)±Δλ_(OL) spectral overlap region about the crossover    wavelengths-   λi/i+1±Δλ_(LK) spectral combination region about the crossover    wavelengths

1. A method for calibrating a photoluminescence measurement system, inparticular a fluorescence measurement system, the method comprising: (a)measuring fluorescence spectra (J_(i)(λ)) of a plurality of fluorescencestandards (i) with the photoluminescence measurement system to becalibrated, wherein the fluorescence standards (i) are selected, so thattheir combined spectrally corrected and normalized fluorescence spectra(I_(i)(λ)) cover a predefined spectral range in such a way, that thefluorescence bands of the spectrally corrected and normalizedfluorescence spectra (I_(i)(λ)) have at least a minimum predefinedintensity at mutual crossover wavelengths (λ_(i/i+1)), wherein thespectrally corrected and normalized fluorescence spectra (I_(i)(λ)) issequentially arranged; (b) calculating combination factors (α_(i))through statistical averaging of consecutive partial correctionfunctions (F_(i)(λ)), wherein the partial correction functions(F_(i)(λ)) are formed by forming the quotient of the measuredfluorescence spectra (J_(i)(λ)) and the corresponding spectrallycorrected and normalized fluorescence spectra (I_(i)(λ)) of thefluorescence standards (i), and wherein the statistical averaging takesplace over a predefined, limited overlap region (λ_(i/i+1)±Δλ_(OL))about the mutual crossover wavelengths (λ_(i/i+1)); and (c) determininga total correction function (F(λ)), for the predefined spectral range bycombination of the partial correction functions (F_(i)(λ)), which areweighted according to the combination factors (α_(i)).
 2. The method ofclaim 1, wherein the spectral overlap region (λ_(i/i+1)±Δλ_(OL);OL=overlap), where the combination factors (α_(i)) are calculated,corresponds to a range of at most ±12 nm about the mutual crossoverwavelengths (λ_(i/i+1)).
 3. The method of claim 1, wherein thefunctional dependence of the total correction function (F(λ)) iscalculated in a predefined, limited combination region(λ_(i/i+1)±Δλ_(LK); LK=linkage) about the mutual crossover wavelengths(λ_(i/i+1)) through statistical averaging of the weighted, consecutivepartial correction functions (F_(i)(λ)).
 4. The method according toclaim 3, wherein the functional dependence of the total correctionfunction (F(λ)) outside the predefined combination region(λ_(i/i+1)±Δλ_(LK)) about the mutual crossover wavelengths (λ_(i/i+1))corresponds to the functional dependence of the weighted, un-averagedpartial correction functions (F_(i)(λ)).
 5. The method according toclaim 3, wherein the predefined combination region (λ_(i/i+1)±Δλ_(LK))about the crossover wavelengths (λ_(i/i+1)) includes ±5 nm about thecorresponding mutual crossover wavelengths (λ_(i/i+1)).
 6. The methodaccording to claim 1, wherein the predefined spectral range of the totalcorrection function (F(λ)) covers at least in part the UV/vis/NIRspectral range.
 7. The method according to claim 1, wherein thepredefined spectral range of a total correction function (F(λ)) forcorrecting the emission extends from 310 to 730 nm and for correctingthe excitation at least from 250 to 630 nm.
 8. The method according toclaim 1, wherein the predefined minimum intensity of the fluorescencebands of the spectrally corrected and normalized fluorescence spectra(I_(i)(λ)) at the mutual crossover wavelengths (λ_(i/i+1)) in theUV/vis/NIR spectral range with λ≦700 nm is at least 20% of a maximumintensity of mutually overlapping flanking bands.
 9. The methodaccording to claim 1, wherein the predefined minimum intensity of thefluorescence bands of the spectrally corrected and normalizedfluorescence spectra (I_(i)(λ)) at the mutual crossover wavelengths(λ_(i/i+1)) in the NIR spectral range with λ>700 nm is at least 10% of amaximum intensity of a mutually overlapping flanking bands.
 10. Themethod according to claim 1, wherein intensities of the spectrallycorrected and normalized fluorescence spectra (I_(i)(λ)) within thespectral overlap region (λ_(i/i+1)±Δλ_(OL)), where the combinationfactors (α_(i)) are calculated, in the UV/vis spectral range with λ≦700nm are at least 10% in relation to maximum intensities of mutuallyoverlapping flanking bands.
 11. The method according to claim 1, whereinthe partial correction functions (F_(i)(λ)) are calculated by dividingthe spectrally corrected and normalized fluorescence spectra (I_(i)(λ))by the corresponding measured fluorescence spectra (J_(i)(λ)), and aspectrum corrected for a blank value is corrected bywavelength-correlated multiplication with the total correction function(F(λ)).
 12. The method according to claim 1, wherein awavelength-dependent combined uncertainty for the predefined spectralrange of total correction function (F(λ)) is computed from respectivecombined uncertainties for individual fluorophores.
 13. A method forcalibrating a photoluminescence measurement system, in particular afluorescence measurement system, the method comprising: (a) measuringfluorescence spectra (J_(i)(λ)) of a plurality of fluorescence standards(i) with the photoluminescence measurement system to be calibrated,wherein the fluorescence standards (i) are selected, so that theircombined spectrally corrected and normalized fluorescence spectra(I_(i)(λ)) cover a predefined spectral range in such a way, that thefluorescence bands of the spectrally corrected and normalizedfluorescence spectra (I_(i)(λ)) have at least a minimum predefinedintensity at mutual crossover wavelengths (λ_(i/i+1)), wherein thespectrally corrected and normalized fluorescence spectra (I_(i)(λ)) issequentially arranged; (b) calculating combination factors (α_(i))through statistical averaging of consecutive partial correctionfunctions (F_(i)(λ)), wherein the partial correction functions(F_(i)(λ)) are formed by forming the quotient of the measuredfluorescence spectra (J_(i)(λ)) and the corresponding spectrallycorrected and normalized fluorescence spectra (I_(i)(λ)) of thefluorescence standards (i), and wherein the statistical averaging takesplace over a predefined, limited overlap region (λ_(i/i+1)±Δλ_(OL))about the mutual crossover wavelengths (λ_(i/i+1)), wherein the spectraloverlap region (λ_(i/i+1)±Δλ_(OL); OL=overlap), where the combinationfactors (α_(i)) are calculated, corresponds to a range of at most ±12 nmabout the mutual crossover wavelengths (λ_(i/i+1)); and (c) determininga total correction function (F(λ)), for the predefined spectral range bycombination of the partial correction functions (F_(i)(λ)), which areweighted according to the combination factors (α_(i)).
 14. A method forcalibrating a photoluminescence measurement system, in particular afluorescence measurement system, the method comprising: (a) measuringfluorescence spectra (J_(i)(λ)) of a plurality of fluorescence standards(i) with the photoluminescence measurement system to be calibrated,wherein the fluorescence standards (i) are selected, so that theircombined spectrally corrected and normalized fluorescence spectra(I_(i)(λ)) cover a predefined spectral range in such a way, that thefluorescence bands of the spectrally corrected and normalizedfluorescence spectra (I_(i)(λ)) have at least a minimum predefinedintensity at mutual crossover wavelengths (λ_(i/i+1)), wherein thespectrally corrected and normalized fluorescence spectra (I_(i)(λ)) issequentially arranged; (b) calculating combination factors (α_(i))through statistical averaging of consecutive partial correctionfunctions (F_(i)(λ)), wherein the partial correction functions(F_(i)(λ)) are formed by forming the quotient of the measuredfluorescence spectra (J_(i)(λ)) and the corresponding spectrallycorrected and normalized fluorescence spectra (I_(i)(λ)) of thefluorescence standards (i), and wherein the statistical averaging takesplace over a predefined, limited overlap region (λ_(i/i+1)±Δλ_(OL))about the mutual crossover wavelengths (λ_(i/i+1)); and (c) determininga total correction function (F(λ)), for the predefined spectral range bycombination of the partial correction functions (F_(i)(λ)), which areweighted according to the combination factors (α_(i)), wherein thefunctional dependence of the total correction function (F(λ)) iscalculated in a predefined, limited combination region(λ_(i/i+1)±Δλ_(LK); LK=linkage) about the mutual crossover wavelengths(λ_(i/i+1)) through statistical averaging of the weighted, consecutivepartial correction functions (F_(i)(λ)).